Synthesis of (2R,3R)-epigallocatechin-3-O-(4-hydroxybenzoate), a novel catechin from Cistus salvifolius, and evaluation of its proteasome inhibitory activities

Article abstract of DOI:10.1016/j.tet.2007.05.043

The total and semi syntheses of (2R,3R)-epigallocatechin-3-O-(4-hydroxybenzoate), a novel catechin from Cistus salvifolius, were accomplished. The proteasome inhibition and cytotoxic activities of the synthetic compound and its acetyl derivative were stud

Full text of DOI:10.1016/j.tet.2007.05.043

Tetrahedron 63 (2007) 7565–7570
Synthesis of (2R,3R)-epigallocatechin-3-O-(4-hydroxybenzoate),
a novel catechin from Cistus salvifolius, and evaluation of its
proteasome inhibitory activities
Kumi Osanai,a Congde Huo,a Kristin R. Landis-Piwowar,b Q. Ping Doub and Tak Hang Chana,
*
aDepartment of Applied Biology and Chemical Technology and the Open Laboratory for Chiral Technology, The Institute of Molecular
Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China
bThe Prevention Program, Barbara Ann Karmanos Cancer Institute, Department of Pathology, School of Medicine,
Wayne State University, Detroit, MI, USA
Received 10 April 2007; revised 10 May 2007; accepted 11 May 2007
Available online 17 May 2007
Abstract—The total and semi syntheses of (2R,3R)-epigallocatechin-3-O-(4-hydroxybenzoate), a novel catechin from Cistus salvifolius,
were accomplished. The proteasome inhibition and cytotoxic activities of the synthetic compound and its acetyl derivative were studied
and compared with (2R,3R)-epigallocatechin-3-gallate (EGCG), the active component from green tea.
Ó 2007 Elsevier Ltd. All rights reserved.
OH
1. Introduction
OH
R
OH
OH
R
The Cistus species are indigenous in the Mediterranean area
and traditionally used in folk medicine for the treatment of
fever, diarrhea, skin diseases, rheumatism, and other inflam-
matory diseases.1 In Turkey, tea prepared from the leaves
of Cistus salvifolius has been used for the treatment of can-
cer.2 While a number of polyphenols have been found within
the genus, the phenolic pattern of Cistus salvifolius L. ap-
peared to differ markedly from other Cistus species.3 The
ethyl acetate-soluble fraction from the aqueous acetone
extract of C. salvifolius was found to contain the following
catechins: catechin (1, C), epicatechin (2, EC), gallocatechin
(3, GC), epigallocatechin (4, EGC), epicatechin-3-gallate (5,
ECG), gallocatechin-3-gallate (6, GCG), epigallocatechin-
3-gallate (7, EGCG), and a novel compound, epigallocate-
chin-3-O-(4-hydroxybenzoate) (8) (Fig. 1). The structure
of 8 was deduced mainly through the 1H NMR of its acetate
(9).3 The 2R,3R absolute configuration in 8 was assigned
solely on the basis of the optical rotation of 9 {[a]2D0 ꢀ60.1
(c 0.06, acetone)}.
O
2
HO
HO
O
2
3
3
O
R1
OH
OH
OH
O
OH
R1
5: 2,3-cis, R=H, R1=OH; ECG
6: 2,3-trans; R=OH, R1=OH; GCG
7: 2,3-cis, R=OH, R1=OH; EGCG
8: 2,3-cis, R=OH, R1=H
1: 2,3-trans; R=H; C
2: 2,3-cis, R=H; EC
3: 2,3-trans; R=OH; GC
4: 2,3-cis, R=OH; EGC
Figure 1. Green tea catechins.
biological activities, which may account for the beneficial
effects attributed to green tea.5,6 Compound 8, on the other
hand, had not been reported to exist in green tea. Regular
drinking of green tea has been associated with reduced
risk of several forms of cancer.7 It is known that a number
of cancer-related proteins are affected by tea polyphenols,
in particular by EGCG. However, the exact mechanism of
tea-mediated cancer prevention is still unknown.7 Recently,
we proposed that inhibition of proteasome may be a key
mechanism in the cancer prevention activity of green tea.8
Furthermore, it is the gallate ester bond-containing tea poly-
phenols (e.g., 5–7) but not the flavan-3-ols (e.g., 1–4), which
inhibit the proteasomal chymotrypsin-like (b5) activities of
the proteasome.9
Tea, produced from the plant Camellia sinensis, has been
consumed by human beings for thousands of years. Tea
catechins constitute about 30–40% of the water-soluble
compounds in brewed green tea. The major tea catechins
are compounds 1–7.4 Of these, EGCG (7) is by far the
most abundant and has been reported to have various
* Corresponding author. Tel.: +852 3400 8670; fax: +852 2364 9932;
e-mail: bcchanth@polyu.edu.hk
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.05.043

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K. Osanai et al. / Tetrahedron 63 (2007) 7565–7570
Because of our interest in the synthesis of EGCG,10 its
analogs,11 and their role as proteasome inhibitors in cancer
protection,12 we became interested in the synthesis of com-
pound 8 and its activity in proteasome inhibition.
DMF/ꢀ60 ꢁC/N2 were found to be the best giving a complex
mixture from which the benzylated derivative 15 could be
obtained in pure form in slightly over 20% after purification
(Scheme 1b). The complex mixture was presumably due to
the formation of both O- and C-benzylated products with
electron-rich phenolic compounds under basic conditions.13
We also tried acid-catalyzed benzylation by using benzyl tri-
chloroacetimidate,14 which was also unsuitable. However,
by converting EGCG to the peracetate 16 in quantitative
yield first,15 we were able to hydrolyze the acetyl function
of 16 and use an in situ benzylation method to form the benz-
ylated compound 15 in a cleaner manner and improved iso-
lated yield (31%). In any case, hydrolysis of compound 15
with sodium hydroxide in methanol gave 13 in 90% yield
(Scheme 1b). Compound 13, obtained in this manner, can
be similarly converted to 8 and 9 as shown in Scheme 1.
2. Results and discussion
2.1. De novo synthesis of compound 8
Previously, we had reported on the enantioselective synthe-
sis of the benzylated epigallocatechin 13 [(ꢀ)-(2R,3R)-5,7-
bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-ol]
starting from 3,5-bis(benzyloxy)phenol (10) and 3,4,5-tris-
(benzyloxy)cinnamyl alcohol (11) (Scheme 1a).10 Acylation
of 13 with 4-(benzyloxy)benzoyl chloride, prepared from
4-benzyloxybenzoic acid and oxalyl chloride, gave the pro-
tected ester 14. Alternately, compound 13 can be acylated
with 4-benzyloxybenzoic acid directly using DCC/DMAP
as the coupling conditions. Hydrogenolysis of 14 afforded
compound 8 in good yield. Acetylation of 8 with acetic
anhydride gave the acetate 9 (Scheme 1), which showed
2.3. Proteasome inhibition of compounds 8 and 9
The eukaryotic proteasome is a large multi-catalytic, multi-
subunit protease complex possessing three distinct activities,
which are associated with three different b subunits, chymo-
trypsin-like (with b5 subunit), trypsin-like (with b2 subunit),
and peptidyl-glutamyl peptide-hydrolyzing-like (PGPH- or
caspase-like; with b1 subunit).16 Inhibition of the chymo-
trypsin-like, but not the trypsin-like, activity of the protea-
some has been found to be associated with induction of
tumor cell apoptosis.17 The discovery of new proteasome
inhibitors with little or no toxicity is highly desirable in
anticancer therapy.18 We have previously reported that
(ꢀ)-EGCG inhibits the chymotrypsin-like activity of the
proteasome in vitro (IC50 86–194 nM) and in intact tumor
cells (1–10 mM)9 leading to accumulation of proteasome
target proteins (such as IkB-a, p27, and Bax) and apoptosis
in human cancer cell lines, as measured by activation of
caspases and cleavage of poly(ADP-ribose) polymerase
(PARP).19 We have now compared the IC50 of EGCG
(0.2 mM) with compound 8 (40.4 mM) for their inhibitory
activity against the chymotrypsin-like activity of a purified
proteasome in vitro (Table 1). It is clear that the loss of the
two hydroxyl groups on the gallate ring has led to much
reduced inhibitory activity (about 200-fold).
1
the same H NMR as those reported for 9 isolated from
C. salvifolius. This confirms the structure of the natural com-
pound 8. Since the total synthesis gave enantioselectively the
2R,3R absolute configuration and since the synthetic 9 has
the same spectroscopic data and identical optical rotation
{[a]2D0 ꢀ60 (c 0.2, acetone)} as the isolated compound 9,
this confirms the stereochemistry of the natural product.
2.2. Semi syntheses of compound 8 from natural EGCG
Even though the above total synthesis was able to give com-
pounds 8 and 9, it involved a number of steps including the
preparation of the starting materials 10 and 11, and the over-
all yield was low (<19% based on 11). This prompted us to
examine the synthesis of 8 from the natural (ꢀ)-EGCG (7),
which is readily available commercially. Although direct
benzylation of EGCG with different benzylating agents
(BnCl, BnBr), different bases (NaH, K2CO3, K2CO3/KI),
and solvents (dimethylformamide (DMF), acetone) at differ-
ent temperatures was tried, the conditions of BnCl/NaH/
OBn
OR
OBn
OBn
OR
OR
OBn
OBn
OBn
BnO
OH
OBn
BnO
OH
RO
O
R
R
BnO
O
a, b, c
d, e, f
g, h
i
S
OBn
(a)
(b)
+
OH
OH
OBn
S
O
OH
OBn
HO
OR
OBn
OBn
10
13
11
12
O
o
OR
OBn
OBn
OBn
14: R=Bn
8: R=H
j
BnO
O
OAc
OH
OAc
OAc
k
OH
OH
9: R=Ac
O
AcO
O
HO
O
OBn
OBn
OBn
l
n
O
15
O
O
OAc
OAc
OAc
OH
OH
OH
OBn
O
O
m
16 (ProE)
7 (EGCG)
OAc
OH
Scheme 1. (a) 25% H2SO4/SiO2; (b) TBSCl, imidazole; (c) AD-mix-a, MeSO2NH2; (d) TBAF; (e) CH(OEt)3, PPTS; (f) K2CO3; (g) Dess–Martin periodinane;
(h) L-Selectride; (i) 4-benzyloxybenzoic acid, DCC, DMAP; (j) Pd(OH)2, H2; (k) Ac2O, DMAP; (l) NaH, BnCl; (m) Ac2O, pyridine; (n) NaH, BnCl, H2O; (o)
NaOH, MeOH.

K. Osanai et al. / Tetrahedron 63 (2007) 7565–7570
Table 1. Inhibition of proteasome activity by EGCG and analogs
7567
inhibit a purified 20S proteasome activity in vitro (Table 1)
but inhibited the proteasome activity in intact tumor cells
in vivo inducing cell death.15 It was reported that 16 was
rapidly converted to EGCG (7) by HCT116 human colon
cancer cells resulting in a 2.8–30-fold greater intracellular
concentration of EGCG as compared to treatment with
EGCG itself.23 These authors further showed that intragas-
tric administration of 16 to CF-1 mice resulted in higher bio-
availability compared to that of equimolar doses of EGCG.23
We have therefore examined the biological activity of com-
pound 9, the acetylated derivative of 8. Interestingly, even
though 9 is not active as an inhibitor of purified proteasome
(Table 1), it showed intracellular proteasome inhibition of
chymotrypsin-like activity (Fig. 2A) in cultured Leukemia
Raji B cells, similar to that of ProE. Proteasome inhibition
was confirmed by the levels of accumulation of ubiquiti-
nated proteins and Bax (Fig. 2B).
Compound
IC50 (mM)
(ꢀ)-EGCG
0.2ꢂ0.02
40.4ꢂ0.17
n.a.a
8
16 (ProE)
9
n.a.a
a
Indicates that the inhibitory activity of the purified proteasome at 25 mM
was <25%.
A critical issue concerning the potential application of
EGCG as an anticancer agent is its known poor bioavailabil-
ity.20 The low bioavailability was thought to be partly due to
the poor stability of EGCG in alkaline or in neutral solu-
tions.21 Another factor may be due to in vivo metabolic
transformation of EGCG into various metabolites.22 To
improve the stability and the potency of (ꢀ)-EGCG, we
recently proposed acetylated EGCG (16, ProE) as a potential
prodrug.15 As prodrugs rationally have low cytotoxicity
prior to the metabolic activation, we found that 16 did not
The acetylated compound 9 induced time-dependent cell
death observed by trypan blue dye exclusion more than the
A
CT-like Activity
120
100
80
60
40
20
0
4h
24h
DMSO
ProE
EGCG
8
9
B
4h
24h
8
9
8
9
Ub
1.0
1.4 1.1
1.6 1.4
1.1 1.4 1.0
1.4 1.5 1.0
2.7 1.2
1.8 1.2
1.3
2.5
Bax
1.0
1.1
1.6
PARP
p85/PARP
Actin
Figure 2. ProE and 9 are proteasome inhibitors in cultured tumor cells. Leukemia Raji B cells were treated, harvested, and analyzed for the chymotrypsin-like
activity, as described in Section 4 (A). Western blot analysis using specific antibodies to ubiquitin, the proteasome target protein Bax, PARP, and actin (B).

7568
K. Osanai et al. / Tetrahedron 63 (2007) 7565–7570
A
(ꢀ)-(2R,3R)-5,7-bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)-
Trypan Blue
phenyl]chroman-3-ol12 (13) and (ꢀ)-(2R,3R)-5,7-diace-
toxy-2-(3,4,5-triacetoxyphenyl)chroman-3-yl-(3,4,5-triace-
toxy)benzoate (16).15
70
60
50
40
30
20
10
0
4h
24h
Anhydrous tetrahydrofuran (THF) was distilled under nitro-
gen from sodium benzophenone ketyl. Anhydrous dichloro-
methane (CH2Cl2) was distilled under nitrogen from CaH2.
Anhydrous DMF was distilled under nitrogen from CaH2.
Reaction flasks were flame-dried under a stream of N2. All
moisture-sensitive reactions were conducted under a nitro-
gen atmosphere. Flash chromatography was carried out us-
ing silica gel 60 (70–230 mesh). The melting points were
uncorrected. 1H and 13C NMR (500 MHz) spectra were mea-
sured with TMS as internal standard when CDCl3 and
CD3OD were used as solvent. High-resolution electrospray
ionization (HRESI) mass spectra were recorded using
a QTOF-2 Micromass spectrometer.
DMSO
EGCG
ProE
8
9
B
Caspase-3 Activity
6
5
4
3
2
1
0
4h
24h
4.1.1. (L)-(2R,3R)-5,7-Bis(benzyloxy)-2-[3,4,5-tris(benz-
yloxy)-phenyl]chroman-3-yl-(4-benzyloxy)benzoate (14)
with (COCl)2, DMAP. To a solution of 4-benzyloxybenzoic
acid (85.1 mg, 373 mmol) in dry CH2Cl2 (7.00 mL) was
added oxalyl chloride (3.00 mL, 34.4 mmol). The mixture
was refluxed at 65 ꢁC for 2 h. After the solution of
the reaction mixture was evaporated, the resulting oil was
dried completely under a reduced pressure for 2 h. The
obtained white solid was dissolved in CH2Cl2 (1.00 mL)
and cooled to 0 ꢁC. 4-Dimethylaminopyridine (DMAP,
55.6 mg, 455 mmol) was added to the solution and the mix-
ture was stirred for 5 min. A solution of (ꢀ)-(2R,3R)-5,7-
bis(benzyloxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-ol
(13, 138 mg, 182 mmol) in dry CH2Cl2 (1.00 mL) was added
dropwise at 0 ꢁC and the mixture was stirred at room temper-
ature overnight. The solvent was evaporated in vacuo and the
resulting oil was purified by flash SiO2 column chromato-
graphy (hexane/EtOAc, 5:1) to give 161 mg (91%) of the title
compound as a pale yellow amorphous solid: [a]2D0 ꢀ42
DMSO
EGCG
ProE
8
9
Figure 3. Acetylated compound 9 induces time-dependent cell death in
cultured tumor cells. Leukemia Raji B cells were treated with the solvent
(DMSO) or 25 mM of the indicated analog for 4 or 24 h, followed by trypan
blue dye exclusion assay. The data represented are the mean number of dead
cells over total cell populationꢂSD after treatment (A). A cell-free caspase-
3/-7 activity assay was performed as described in Section 4. Caspase-3/-7
activity is represented as a percentage of the solvent (DMSO) control (B).
parent compound 8 and EGCG, though not quite to the same
extent as ProE (Fig. 3A). To determine whether the observed
cell death (Fig. 3A) was due to apoptosis, the cell extracts
from the same experiments were used for measurement
of caspase-3 activation (Fig. 3B) and PARP cleavage
(Fig. 2B). Indeed, after 24 h treatment, compound 9 induced
nearly 4.3-fold increase in caspase-3 activity in Raji B cells
compared with the control experiment. These results indi-
cate that the acetylated compound 9 is more potent than
either its parent compound 8 or EGCG itself in inhibiting
proteasome activity and inducing apoptotic cell death in
a time-dependent manner.
1
(c 0.70, CHCl3); H NMR (CDCl3) d 7.87 (d, J¼7.5 Hz,
2H), 7.40–7.10 (m, 30H), 6.84 (d, J¼7.5 Hz, 2H), 6.71 (s,
2H), 6.27 (br s, 1H), 6.23 (br s, H), 5.59 (br s, 1H), 5.01–
4.85 (m, 10H), 4.72–4.67 (m, 2H), 3.10–2.97 (m, 2H); 13C
NMR (CDCl3) d 165.3, 162.9, 159.0, 158.2, 155.8, 153.1,
138.5, 138.1, 137.3, 137.1, 137.1, 136.3, 133.6, 132.1, 128.9,
128.9, 128.8, 128.6, 128.4, 128.3, 128.3, 128.2, 128.0, 128.0,
127.8, 127.8, 127.7, 127.7, 127.4, 122.9, 114.7, 106.9, 101.2,
95.0, 94.2, 78.1, 75.3, 71.4, 70.4, 70.3, 70.2, 68.2, 26.4;
LRMS (ESI) m/z 989 (M+Na); HRMS m/z calculated for
C64H54O9Na (M+Na) 989.3666, found 989.3616.
3. Conclusion
In conclusion, we have confirmed by synthetic studies the
structure and the absolute configurations of a novel catechin
(2R,3R)-epigallocatechin-3-O-(4-hydroxybenzoate) (8) iso-
lated from C. salvifolius. Compound 8 is less potent than
EGCG in proteasome inhibition in vitro. However, its acetyl
derivative 9 was found to be nearly comparable to ProE (16)
in inducing tumor cell death by apoptosis.
4.1.2. Benzylated (L)-EGCG, [(L)-(2R,3R)-5,7-bis(benz-
yloxy)-2-(3,4,5-tris(benzyloxy)phenyl)chroman-3-yl-
(3,4,5-trisbenzyloxy)benzoate (15)]. Method 1: to a solu-
tion of EGCG (7, 458 mg, 1.00 mmol) in dry DMF
(10.0 mL) under nitrogen at ꢀ60 ꢁC, NaH (60% dispersion
in mineral oil, 200 mg, 5.00 mmol) was added. After 2 h,
benzyl chloride (920 mL, 8.00 mmol) was added via a sy-
ringe. The mixture was stirred at ꢀ60 ꢁC for an additional
30 min and then at room temperature for 24 h. The reaction
was quenched by adding hydrochloric acid (1 N, 2.00 mL)
and water (15.0 mL). The aqueous layer was extracted
with EtOAcꢃ3. The combined organic layers were washed
with water and brine, then dried over sodium sulfate
4. Experimental
4.1. General
The starting materials and reagents, purchased from com-
mercial suppliers, were used without further purification.
Literature procedures were used for the preparation of

K. Osanai et al. / Tetrahedron 63 (2007) 7565–7570
7569
(Na2SO4), and evaporated in vacuo. The residue was purified
by flash SiO2 column chromatography (hexane/EtOAc, 5:1)
to afford the crude benzylated EGCG as pale yellow oil.
After precipitation of the crude mixture by mixed-solvent
(hexane/EtOAc/dichloromethane), 248 mg (21%) of the title
compound was obtained as a white solid.
by flash SiO2 column chromatography (hexane/EtOAc, 5:1)
to afford 72.5 mg (75%) of the desired compound as a pale
yellow amorphous solid. The spectroscopic data of the com-
pound were identical to that of compound 14 obtained
above.
4.1.5. (2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxyphen-
yl)chroman-3-yl-40-hydroxybenzoate (8). Under a hydro-
gen atmosphere, palladium hydroxide on carbon powder,
20% Pd (5.00 mg) was added to a solution of (2R,3R)-5,7-
bis(benzyloxy)-2-[(3,4,5-tris (benzyloxy)phenyl]chroman-
3-yl-(40-benzyloxy)benzoate (14, 48.4 mg, 50.0 mmol) in
EtOAc (5.00 mL). The resulting mixture was stirred at
room temperature until TLC showed that the reaction was
completed. Then the mixture was filtered to remove the cat-
alyst. The filtrate was evaporated in vacuo to afford 20.4 mg
(96%) of the title compound as a white foam solid: [a]2D0 ꢀ57
Method 2: to a mixture of (ꢀ)-(2R,3R)-5,7-diacetoxy-
2-(3,4,5-triacetoxyphenyl)chroman-3-yl-(3,4,5-triacetoxy)-
benzoate (16, 794 mg, 1.00 mmol), benzyl chloride (920 mL,
8.00 mmol), NaH (60% dispersion in mineral oil, 200 mg,
5.00 mmol), and DMF (20.0 mL), water (144 mL, 8.00 mmol)
was added at 0 ꢁC dropwise over a period of 10 min. After
stirring overnight at room temperature, the reaction mixture
was diluted with EtOAc and washed with water and brine,
then dried over Na2SO4, and evaporated in vacuo. The resi-
due was purified by flash SiO2 column chromatography (hex-
ane/EtOAc, 5:1) and subsequently precipitation by hexane/
EtOAc/dichloromethane to afford 366 mg (31%) of the title
1
(c 0.23, MeOH); H NMR (CD3OD) d 7.78 (d, J¼8.0 Hz,
2H), 6.83 (d, J¼8.0 Hz, 2H), 6.63 (s, 2H), 6.05 (d,
J¼2.0 Hz, 1H), 6.03 (d, J¼1.5 Hz, 1H), 5.54 (m, 1H), 5.09
(s, 1H), 3.05 (dd, J¼17.0, 4.5 Hz, 1H), 2.95 (dd, J¼17.0,
2.5 Hz, 1H); 13C NMR (CD3OD) d 165.3, 161.9, 157.1,
156.8, 156.4, 145.6, 131.9, 130.1, 121.9, 115.3, 105.9,
98.2, 95.7, 95.0, 77.4, 68.9, 25.8; LRMS (ESI) m/z 449
(M+Na); HRMS m/z calculated for C22H18O9H (M+H)
427.1029, found 427.1028.
1
compound as a white solid: [a]2D0 ꢀ44 (c 2.0, CHCl3); H
NMR (CDCl3) d 7.42–7.18 (m, 42H), 6.73 (s, 2H), 6.39 (d,
J¼2.0 Hz, 1H), 6.34 (d, J¼2.0 Hz, 1H), 5.66 (s, 1H), 5.05–
4.90 (m, 13), 4.79 (d, J¼11.0 Hz, 1H), 4.67 (d, J¼11.0 Hz,
1H), 3.12 (dd, J¼17.5, 4.5 Hz, 1H), 3.06 (dd, J¼17.5,
2.5 Hz, 1H); 13C NMR (CDCl3, 125 MHz) d 165.0, 159.1,
158.3, 155.9, 153.1, 152.6, 143.0, 138.7, 138.0, 137.7, 137.1,
137.0, 137.0, 136.7, 133.5, 128.8, 128.8, 128.8, 128.7, 128.6,
128.5, 128.4, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.0,
128.0, 127.7, 127.7, 127.5, 125.2, 109.4, 107.0, 101.2, 94.9,
94.2, 78.2, 75.4, 75.3, 71.4, 71.3, 70.4, 70.3, 68.5, 26.5;
LRMS (ESI) m/z 1201 (M+Na); HRMS m/z calculated for
C78H66O11Na 1201.4503, found 1201.4519.
4.1.6. (2R,3R)-5,7-Diacetoxy-2-(3,4,5-triacetoxyphenyl)-
chroman-3-yl-40-acetoxybenzoate (9). To a solution of
(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-
3-yl-40-hydroxybenzoate (8 (17.1 mg, 40.0 mmol) and Ac2O
(28.4 mL, 300 mmol) in dry MeCN (5.00 mL) at ꢀ40 ꢁC,
DMAP (5.00 mg, 40.0 mmol) was added. The resulting mix-
ture was stirred for 1 h. Saturated NaHCO3 aqueous solution
was added and the mixturewas allowed towarm to room tem-
perature. The mixture was extracted with EtOAcꢃ3 and the
combined organic phases were washed with water and brine,
dried over Na2SO4, and evaporated invacuo. The residue was
purified by flash SiO2 column chromatography (hexane/
EtOAc, 1:1) to afford 24.4 mg (90%) of the title product as
a white solid: Mp 109–111 ꢁC; [a]D20 ꢀ60 (c 0.2, acetone);
1H NMR (CDCl3) d 7.87 (d, J¼8.0 Hz, 2H), 7.27 (s, 2H),
7.09 (d, J¼8.0 Hz, 2H), 6.74 (d, J¼2.0 Hz, 1H), 6.59 (d,
J¼2.0 Hz, 1H), 5.62 (s, 1H), 5.20 (s, 1H), 3.06 (m, 2H),
2.28 (s, 6H), 2.27 (s, 3H), 2.24 (s, 3H), 2.23 (s, 6H); 13C
NMR (CDCl3) d 168.9, 168.6, 168.4, 167.5, 166.7, 165.0,
154.7, 154.4, 149.7, 149.7, 143.4, 135.4, 134.2, 131.4, 126.9,
121.5, 118.6, 109.5, 108.9, 108.0, 76.4, 67.5, 25.9, 21.1, 20.7,
20.6, 20.1; LRMS (ESI) m/z 701 (M+Na); HRMS m/z calcu-
lated for C34H30O15Na 701.1482, found 701.1472.
4.1.3. Benzylated (L)-EGC, [(L)-(2R,3R)-5,7-bis(benzyl-
oxy)-2-[3,4,5-tris(benzyloxy)phenyl]chroman-3-ol (13)].
To a solution of (ꢀ)-(2R,3R)-5,7-bis(benzyloxy)-2-(3,4,5-
tris(benzyloxy)phenyl)chroman-3-yl-(3,4,5-tris)benzyloxy)-
benzoate (15, 236 mg, 200 mmol) in MeOH (15.0 mL),
sodium hydroxide (8.00 mg, 200 mmol) was added. The
resulting mixture was stirred at room temperature for 4 h,
concentrated and water was added, then the mixture was ex-
tracted with EtOAcꢃ3. The combined organic layers were
dried over Na2SO4 and evaporated in vacuo. The residue
was purified by Flash SiO2 column chromatography (hex-
ane/EtOAc, 4:1) to afford 136 mg (90%) of the title com-
pound as a white solid. The spectroscopic data of the
compound were identical to that of compound 13 reported
in the literature.
4.1.4. (2R,3R)-5,7-Bis(benzyloxy)-2-[(3,4,5-tris(benzyloxy)-
phenyl]chroman-3-yl-(4-benzyloxy)benzoate (14) with
DCC, DMAP. To a solution of 4-benzyloxylbenzoic acid
(34.2 mg, 150 mmol) in dry CH2Cl2 (10.0 mL), dicyclo-
hexylcarbodiimide (DCC, 31.0 mg, 150 mmol) was added.
The resulting mixture was stirred at room temperature for
4 h and cooled to 0 ꢁC. DMAP (1.20 mg, 100 mmol.) was
added and then a solution of (2R,3R)-5,7-bis(benzyloxy)-2-
[3,4,5-tris(benzyloxy)phenyl]chroman-3-ol (13, 75.7 mg,
100 mmol) in dry CH2Cl2 (2.00 mL) was added dropwise.
The mixture was refluxed for 24 h and concentrated. After
EtOAc (1.00 mL) was added and the mixture was cooled
in fridge, the urea byproduct was filtered, and the filtrate
was evaporated in vacuo. The resulting residue was purified
4.2. Cell culture
Human Raji B cells were cultured in RPMI supplemented
with 10% (v/v) fetal bovine serum, 100 U/mL penicillin,
and 100 mg/mL streptomycin. Cell cultures were maintained
in a 5% CO2 atmosphere at 37 ꢁC.
4.3. Inhibition of purified 20S proteasome activity
Measurement of the chymotrypsin-like activity of the 20S
proteasome was performed by incubating 35 mg of purified
rabbit 20S proteasome with 40 mM of fluorogenic peptide

7570
K. Osanai et al. / Tetrahedron 63 (2007) 7565–7570
substrate, Suc-Leu-Leu-Val-Tyr-AMC, with or without a tea
polyphenol at various concentrations, as described previ-
ously.9
References and notes
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4.4. Cell extract preparation and western blotting
Whole cell extracts were prepared as described previously.9
Analysis of Bax, PARP, and ubiquitinated proteins was per-
formed using monoclonal or polyclonal antibodies, accord-
ing to previously reported protocols.9 Densitometry was
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tumor cells
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Cells were treated with each compound at 25 mM for 4 or
24 h, harvested, and lysed as described previously. Whole
cell extracts (10 mg) were incubated with Suc-Leu-Leu-
Val-Tyr-AMC (40 mM) fluorogenic substrate at 37 ꢁC in
100 mL of assay buffer (50 mM Tris–HCl, pH 8) for 2.5 h.
After incubation, production of hydrolyzed 7-amino-4-
methylcoumarin (AMC) groups was measured using a Victor
3 Multilabel Counter with an excitation filter of 380 nm and
an emission filter of 460 nm (Perkin–Elmer, Boston, MA,
USA).
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The trypan blue dye exclusion assay was used to ascertain
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4.7. Induction of caspase-3 activity
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Cells were treated with each compound at 25 mM for 4 or
24 h, harvested, and lysed as described previously.9 Ac-
DEVD-AMC (40 mM) was then incubated with the prepared
cell lysates for 2.5 h and the caspase-3 activity was measured
as described previously.21,22
16. Schwartz, A. L.; Ciechanover, A. Ann. Rev. Med. 1999, 50, 57.
€
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Acknowledgements
We thank the Areas of Excellence Scheme established under
the University Grants Committee of the Hong Kong Admin-
istrative Region, China (Project No. AoE/P-10/01) for finan-
cial support. Funding support by National Cancer Institute
(Grant Number: 1R01CA120009 and 5R03CA112625) is
gratefully acknowledged.
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Chem. 2001, 49, 477.
22. Lu, H.; Meng, X.; Yang, C. S. Drug Metab. Dispos. 2003,
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C. T.; Yang, C. S. Drug Metab. Dispos. 2006, 34, 2111.